| Background:Hepatic fibrosis is a frequent consequence of endogenous and exogenous pathogenic factors that attack liver continuously, such as viruses, bacteria and Parasites infection, bile duct obstruction, some chemical drugs, poisons and lipid droplet deposition. The basic pathological changes of hepatic fibrosis is charactered by deposition of extracellular matrix(ECM) produced by hepatic stellate cell(HSC).Hepatic fibrosis is the common pathway of liver cirrhosis and hepatocellular carcinoma leading to human death.Smad3 signal pathway is most closely related to activation of HSC. Smad3 is the key molecule in this pathway and plays an important role in HSC activation. In previous studies, we have found that Smad3 signal pathway is involved in the formation and development of hepatic fibrosis in rats. Once the expression of Smad3 was down-regulated, the content of ECM was reduced in the liver.The present studies show that hepatic fibrosis can be reversed when HSC activation is inhibited and ECM is reduced. Silybin capsule is a classic drug treating liver diseases and can repair liver damage. Silybin is the main ingredient. Silybin has an effective antioxidant and inhibits lipid peroxidants. It also plays an important role,such as improving liver detoxification capacity, maintainingliquidity of cell membrane, protecting the membrane of hepatocytes, promoting repairation of hepatocytes and reversing fibrogenesis. However it has been unclear whether Smad3 signal pathway is involved in the protective effects of silybin on hepatic fibrosis TAA-induced in Beagles.Objectives:To investigate the protective effect of silybin on TAA-induced hepatic fibrosis in Beagles, to explore whether Smad3 signal pathway is involved in the process.Methods:Eighteen healthy male Beagles was randomly assigned to three groups of 6 each.Beagles in nomal group were given neither TAA nor silybin and had a free access to food and water for twelve weeks; Beagles were subcutaneous injection of TAA(12mg/kg, two times per week) in model group lasting for 12 weeeks;In TAA/silybin group, Beagles were subcutaneous injection of TAA(12mg/kg, two times per week) in model group lasting for 12 weeeks and simultaneously each Beagle was given silybin(28mg/kg/d) at week 5.At week 12, all the Beagles were narcotized with pentobarbital sodiumsolution through intravenous injection, and then liver tissues were collected from the same liver lobe and were fixed. HE staining can observe morphological changes of liver tissue and collagen fiber content in liver tissue were detected by Masson staining. The expression and distribution of alpha-smooth muscle actin(α-SMA), Collagen Iand Smad3 of hepatic tissues was examined by immunohistochemistry.Results:1. The HE stainingshowed that intact lobular architecture was observed in normal group and hepatocytes was in order. Hepatocytes are uniform, full of cytoplasm. In model group there was disordered organizational structure of liver, hepatocytes swelling, fibrous connective tissue formation and inflammatory cells infiltration. In TAA /silybin group, pathological changes reduced significantly compared with that in model group. However, the changes were serious compared with that in nomal group.2. Compared with that in normal group, there was more fibril protein in model group,collagen area percentage was higher( 12.77 ±0.43 vs 0.30 ±0.02, P<0.01). In TAA/silybin group it was lower than that in model group(4.68 ±0.22 vs 12.77 ±0.43, P<0.01), but it was higher than that in normal group( 4.68±0.22 vs 0.30±0.02, P<0.01).The difference was statistically significant.3.The analysis of immunohistochemistry was as follows.The brown yellow and yellow particles in the images were determined the positive expression of immunohistochemistry results.3.1 The expression of α-SMA: in nomal group, α-SMA expressed rarely in liver portal area; in model group, a lot of brown particles distributed in the liver portal area and fiber interval; in TAA/silybin group, α-SMA expression was decreaed.3.2 The expression of Collagen I: in nomalgroup, collagen I, pale yellow filaments, was only expressed around the vascular wall and bile duct wall; in model group, a lot of heavy brown fiber was observed around the liver portal area and fibrosis area; in TAA/silybin group, Collagen I expression was decreaed and it was lighter than that in model group.3.3 The expression of Smad3: Smad3 expression could be observed in perisinusoidal space and hepatic cytoplasm, which was light yellow; in model group, Smad3 xpression presented larger and deeper colored particles around the liver portal rea, perisinusoidal space and liver cells; in TAA/ silybin group, the expression of Smad3 showed a significant decrease.These images were analyzed by Image-Proplus 6.0 semi-quantitative analysis software. The results showed: the expressions of α-SMA、Collagen Iand Smad3 in model group significantly increased compared with that in normal group,( 15.93±1.59vs1.52±0.17; 20.19±0.15 vs 0.41±0.02; 6.69±0.23 vs 0.90±0.09, P <0.01). These data in TAA/silybin group were lower than that in TAA model group(5.39±0.31 vs 15.93±1.59; 5.04±0.09 vs 20.19±0.15; 2.34±0.31 vs 6.69±0.23, P <0.01), but higher than that in nomal group(5.39±0.31 vs 1.52±0.17; 5.04±0.09 vs0.41±0.02, P < 0.01; 2.34±0.31 vs 0.90±0.09, P < 0.05). The differences were statistically significant.Conclusions:Silybin can attenuate TAA-induced hepatic fibrosis in Beagles via inhibiting activation of HSC and reducing ECM production. Smad3 signal pathway may beinvolved in this process. |
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